PROJECT SUMMARY/ABSTRACT Our recent studies have demonstrated that abnormal activation of the Wnt pathway plays key roles in pathogenesis of retinal inflammation, NV and fibrosis in both AMD and diabetic retinopathy. A genetic study has reported that variants of very low-density lipoprotein receptor (VLDLR) are associated with AMD. We have recently reported that VLDLR functions as a negative regulator of the Wnt pathway, as VLDLR knockout resulted in Wnt pathway over-activation, leading to AMD-like pathologies, such as retinal inflammation, vascular leakage and sub-retinal NV. Toward the mechanism by which VLDLR regulates the Wnt pathway, we have recently obtained the following preliminary data: 1) Expression of VLDLR and its soluble extracellular domain (VLDLRN) inhibits Wnt signaling. 2) The VLDLRN peptide decreases total LRP6 protein levels. 3) Co-immunoprecipitation assay showed that VLDLRN binds with LRP6, forming a VLDLR-LRP6 heterodimer. 4) VLDLRN peptide blocks Fz-LRP6 dimerization induced by Wnt ligand. Based on these observations, we hypothesize that VLDLR forms a heterodimer with LRP6, which blocks the Wnt ligand-induced LRP6 aggregation and LRP6 signalosome formation. This binding may represent a mechanism by which VLDLR inhibits Wnt signaling. To test this hypothesis, we will determine if binding of VLDLR to LRP6 blocks the Wnt ligand-induced LRP6 aggregation and formation of signalosomes. We will also determine if binding of VLDLR to LRP6 affects LRP6 endocytosis and stability. Further, we will define the sequence domains responsible for the interaction between VLDLR and LRP6 using deletion mutants and co-immunoprecipitation assays. The therapeutic potential of the sequence domain of VLDLR binding to LRP6 and inhibiting Wnt signaling will be explored by evaluating its efficacy on retinal inflammation, vascular leakage and NV. Recently, two independent large clinical trials reported that fenofibrate, a PPAR agonist which lowers VLDL levels in the circulation, has therapeutic effects on retinal vascular leakage and NV in type 2 diabetic patients. Our preliminary studies found that fenofibrate inhibits Wnt signaling and up- regulates VLDLR expression and its promoter activity. Therefore, we will test the hypothesis that inhibition of Wnt signaling through up-regulation of VLDLR expression by fenofibrate represents a mechanism for its beneficial effects on retinal inflammation, vascular leakage and NV. We will use VLDLR KO mice, PPAR KO mice and primary RPE and endothelial cells from these KO mice to determine if VLDLR and PPAR are essential for mediating the Wnt-inhibiting effect of fenofibrate. These studies will elucidate the mechanism by which VLDLR regulates the Wnt pathway and identify a novel, endogenous regulatory mechanism for this important pathway. The proposed studies will reveal the interactions between PPAR and the canonical Wnt pathway and contribute to the development of new treatment for AMD.